Thai J. Pharm. Sci. 34 (2010) 1-19 1Review article Biopharmaceuticals: an overview Bhupinder Singh Sekhon Institute of Pharmacy, Punjab College of Technical Education, Jhande (Ludhiana)-142 021, India Corresponding author. E-mail address: firstname.lastname@example.org Abstract: Biopharmaceuticals drugs structurally mimics compounds found within the body and are produced using biotechnologies. These have the potential to cure diseases rather than merely treat symptoms, and have fewer side effects because of their specificity, for example, cytokines, enzymes, hormones, clotting factors, vaccines, monoclonal antibodies, cell therapies, antisense drugs, and peptide therapeutics. Emerging technologies in the area of biopharmaceuticals include manufacture of monoclonal antibodies in protein free media, designing chemically defined cells, genome based technologies, improving vaccine manufacturing processes, a potential cancer treatment and non-ribosomal peptide synthesis. Biopharmaceuticals have changed the treatment ways of many diseases like diabetes, malignant disorders; since these can be tailored for specific medical problems in different individuals. With biotechnology, any drug can be genetically modified using cell fusion or deoxyribonucleic acid (DNA)-recombinant technologies to alter specificities for individual diseases. Some distinct advantages of biotechnological processes include fewer side effects and more potent effect on target cells. Biopharmaceuticalsû greatest potential lies in gene therapy and genetic engineering. Keywords: Biopharmaceuticals; Bioprocessing; Biotechnology; Transgenics
2 B.S. SekhonIntroduction of genetic information of all living material have already Biopharmaceuticals make up about one-third of developed into an autonomous discipline. The biopharmaceuticaldrugs currently in development and refer to pharmaceutical industry is in a growth phase and is greatly changingsubstances derived from biological sources. These are the way that drugs are produced-from the use of chemicalmedical drugs produced using biotechnology especially synthesis (traditional pharmaceuticals) to biomanufacturinggenetic engineering or hybridoma technology or via (biologics). ùQuality by designû requires a thoroughbiopharmaceutical techniques such as recombinant understanding of a biopharmaceutical product and itshuman technology, gene transfer and antibody production manufacturing processes, necessitating an investment inmethods. Virtually all biotherapeutic agents in clinical time and resources upfront in the discovery anduse are biotech pharmaceuticals. Alternatively, any development of a product . The aims of developmentmedically useful drug whose manufacture involves of a biopharmaceutical are: it should be clinically effective,microorganisms or genetically modified organism or approvable by regulatory authorities and commercially viable.substances that living organisms produce (e.g. enzymes), Development of new drugs and vaccines viaor bioprocessing is termed a biopharmaceutical [1, 2]. biopharmaceutical research require concentrated effortsBiopharmaceutical drugs are large, complex protein on many levels, as well as multiple skills and expertise.molecules derived from living cells. Manufacturing of Various technologies such as manufacture of monoclonalpharmaceutical proteins including antibodies has been antibodies in protein free media; designing chemicallyreported on a large scale. The production systems defined cells, genome based technologies, improvingavailable include mammalian cells, yeast, insect cells vaccine manufacturing processes, a potential cancerand bacteria, and the schematic production work treatment and non-ribosomal peptide synthesis [4,5]flows of important product groups are given in Figure 1. developed in the last decade function similarly to unitThe choice of production systems depends on the nature operations for producing advanced biopharmaceuticalsof the protein being produced. However, there is no . The biopharmaceutical industry is the most importantprecise scientific definition of a biopharmaceutical. sector in industrial biotechnology, and is one of the Biopharmaceuticals, outcome of the exploitation most rapidly growing high-tech industries [7, 8]. Bacteria Multi-domain fusion proteins Yeast Monoclonal antibody DNA Viral or bacterial coat protein Mammalian cell Insect cell Enzyme Examples Recombinant protein Genetically modified cell Production of protein in a cell Purification Recombinant protein Cell therapy Cells of animal or human origin Isolation of cells Expansion Purification Cells for implantation Vaccine against viral infection Production of carrier system Inoculation with virus Elimination of reproducibility and infectivity Purification Virus fragmentFigure 1 Schematic production work flows of important product groups (For example, recombinant protein, cells for implantation, virus fragment)
Thai J. Pharm. Sci. 34 (2010) 1-19 3 Biopharmaceuticals are proteins (including antibodies), biological activity requirements . Biopharmaceuticalsnucleic acids (DNA, RNA or antisense oligonucleotides) have more potential heterogeneity than small moleculeused for therapeutic or in vivo diagnostic purposes, drugs. The large majority of biopharmaceutical productsand are produced by means other than direct extraction are derived from life forms. Small molecule drugs arefrom a native (non-engineered) biological source. The not typically regarded as biopharmaceutical in naturekey areas of investigation in the field, covers drug production, by the industry. The nature of the manufacturing process,plus the biochemical and molecular mechanisms of action and the safety and efficacy profile of biopharmaceuticaltogether with the biotechnology of major biopharmaceutical products are also different.types on the market or currently under development . The majority of first generation biopharmaceuticalsThe first biopharmaceutical substance approved for are unengineered murine monoclonal antibodies or simpletherapeutic use was biosynthetic ùhumanû insulin made replacement proteins displaying an identical aminovia recombinant DNA technology in 1982. In the late acid sequence to a native human protein. Modern1990s advances in manufacturing and processing biopharmaceuticals are engineered, second-generationrevolutionized the production of biopharmaceuticals products. Engineering can entail alteration of amino acidsuch as recombinant DNA technology and hybridoma sequence, glycocomponent of a glycosylated protein,technology. In other words, biopharmaceuticals have or the covalent attachment of chemical moieties suchrevolutionized the treatment of many diseases like as polyethylene glycol. Engineering has been applieddiabetes, malignant disorders etc. More than 150 biotech in order to alter immunological or pharmacokineticdrugs (human insulin, interferons, human growth hormones profile of protein, or in order to generate novel fusionand monoclonal antibodies, as well as thirteen blockbuster products .drugs) are currently marketed around the world .The biopharma market is growing at an annual rate of Biopharmaceutical classification systemaround 15%-far higher than pharmaceuticals (c.6-7% Biopharmaceutical classification system (BCS) isper annum). In future, the market is forecast to be significantly a drug development tool that deals with the contributionsdriven by a shift in usage from conventional drugs to of three major factors, dissolution, solubility and intestinalbiopharma products . Majority of biopharmaceuticals permeability, affecting oral drug absorption fromproducts consist of glycoproteins and methods are immediate release solid oral dosage forms. Accordingnow becoming available that allow the production of to BCS, drug substances are classified into differentrecombinant monoclonal antibodies bearing pre-selected classes . Class I: high solubility-high permeability;oligosaccharides-glycoforms-to provide maximum efficacy Class II: low solubility-high permeability; Class III: highfor a given disease indication . solubility-low permeability; Class IV: low solubility-low permeability.Biopharmaceuticals versus conventional chemicaldrugs Types of biopharmaceuticals Biopharmaceuticals are fundamentally different Biopharmaceuticals are being developed to fightfrom the conventional small molecule chemical drugs cancer, viral infections, diabetes, hepatitis and multiple. There is a fundamental difference in the average sclerosis and these can be grouped into varioussize of the two types of drugs. The chemically synthesized categories. i) cytokines ii) enzymes iii) hormones iv)products are known as çsmall moleculesé drugs (e.g. clotting factors v) vaccines vi) monoclonal antibodiesaspirin, molecular weight 180 Da). In general, the vii) cell therapies viii) antisense drugs, and ix) peptidebiopharmaceuticals are complex macromolecules that therapeutics.are over 100 times larger (e.g. interferon beta, molecular i) Cytokines: Cytokines are hormone-like moleculesweight 19,000 Da) with complex structural and appropriate that can control reactions between cells. They activate
4 B.S. Sekhoncells of the immune system such as lymphocytes and as uric acid crystals or ATP) [27, 28]. Thus, they aremacrophages . Interferon is potent glycoprotein considered major mediators of innate immune reactionscytokine that acts against viruses and uncontrolled cell and blockade of IL-1 by the interleukin-1 receptorproliferation . Interleukins function as messengers antagonist (IL-1RA) has proven a central role of IL-1αfor various steps in the immune process. or IL-1β in a number of human auto-inflammatory diseases Interleukin-2 (IL-2): IL-2 stimulates T lymphocytes. [29-31].The FDA has approved a recombinant variant of IL-2, The signaling of the founding members, IL-1α andaldesleukin (Proleukin), for treating renal cell carcinoma IL-1β, share only 24% amino-acid sequence identity. The antitumor effect of IL-2 and its recombinant but have largely identical biological function . Further,variant was found directly proportional to amount of IL-1 pathway has been reported . IL-1 is made mainlythe agent administered. Endogenous IL-2 is scarce; by one type of white blood cell, the macrophage, andaldesleukin can be mass-produced but has adverse helps another type of white blood cell, the lymphocyte,side effects at relatively low levels of administration . to fight infections. It also helps leukocytes pass throughThe mechanism of action, methods of delivery, efficacy, blood vessel walls to sites of infection and causesand side effect profile of the cytokines IL-2 and interferon fever by affecting areas of the brain that controls bodyalfa were reported . temperature. Interleukin-3 (IL-3): IL-3 is an interleukin, a type of Interleukin 1β (IL-1β): IL-1β is a potent proinflammatorybiological signal (cytokine) that can improve the bodyûs factor during viral infection. Interleukin-1 made in thenatural response to disease as part of the immune laboratory is used as a biological response modifiersystem. It acts by binding to the Interleukin-3 receptor. to boost the immune system in cancer therapy .IL-3 stimulates bone marrow stem cells. Stimulation An IL-1 blocker, anakinra (Kineret), has been approvedof hematopoietic IL-3 and granulocyte-macrophage for treatment of rheumatoid arthritis. Another, rilonaceptcolony-stimulating factor (GM-CSF) appears to be able (Arcalyst, has been approved for cryopyrin-associatedto stimulate sympathetic nerve growth, via specific periodic syndromes .cytokine receptors on neurons, which lead to activation Inflammasome: The inflammasome is a multiproteinof the mitogen-activated protein (MAP) kinase pathway complex that mediates the activation of caspase-1, whichthat then mediated the observed neurotrophic effects promotes secretion of the proinflammatory cytokines. Researchers suggested that IL-3 neuroprotected interleukin 1β (IL-1β) and IL-18, as well as ùpyroptosisû,neuronal cells against neurodegenerative agents like a form of cell death induced by bacterial pathogens.amyloid-β protein (Aβ) . Members of the Nod-like receptor family, including Interleukin-1 (IL-1): A protein produced by various NLRP1, NLRP3 and NLRC4, and the adaptor apoptosis-cells, including macrophages. IL-1 raises body temperature, associated speck-like protein containing a C-terminalspurs the production of interferon, and stimulates growth caspase recruitment domain (ASC) are critical componentsof disease-fighting cells, among other functions. The of the inflammasome that links microbial and endogenousIL-1 family of cytokines comprises 11 proteins (IL-1F1 ùdangerû signals to caspase-1 activation. Several diseasesto IL-1F11) encoded by 11 distinct genes (IL1A, IL1B, are associated with dysregulated activation of caspase-1IL1RN, IL18, and IL1F5 to IL1F11 in man, Il1A to Ilf11 and secretion of IL-1β. In view of above, understandingin mice) [24-26]. inflammasome pathways may provide insight into disease The main function of IL-1-type cytokines is to control pathogenesis that might identify potential targets forproinflammatory reactions in response to tissue injury therapeutic intervention . Inflammasomes and IL-1by pathogen-associated molecular pattern (such as bacterial are involved in the pathogenesis of several inflammatoryor viral products) or damage-or danger-associated disorders. The remarkable progress in this field hasmolecular patterns released from damaged cells (such offered new hope for many patients with these disorders
Thai J. Pharm. Sci. 34 (2010) 1-19 5and also highlighted the role IL-1 might have in other Inhibiting this protein is highly efficient in blockinginflammatory disorders, such as systemic juvenile virus replication .idiopathic arthritis (sJIA), adult-onset Stillûs disease vi) Monoclonal antibodies: Monoclonal antibodies(AOSD), and rheumatoid arthritis . are produced from immortal cells with an antibody Granulocyte-colony stimulating factor (G-CSF) producing spleen cells. Examples include Infliximabstimulates the bone marrow to produce neutrophils (Remicade), adalimumab (Humira), rituximab (Rituxan(antibacterial leukocytes), and is used for cancer , MabThera). Monoclonal antibodies now account fortreatments that are immunodepressants . approximately one third of all new treatments. Their Granulocyte-macrophage colony-stimulating factor applications include the treatment of breast cancers,(GM-CSF) stimulates the bone marrow to produce leukemia, asthma, rheumatoid arthritis, psoriasis, chronicneutrophils and macrophages, and is used for chemo gastrointestinal inflammatory disease and transplantand radio therapy that suppresses bone marrow function rejection. First fully human monoclonal antibody was. launched in 2003 (Humira) in UK-removing potential ii) Enzymes: These are complex proteins that cause for immunogenic reactions. New indications and therapiesa specific chemical change in other substances without are emerging all the time. The development of humanbeing changed themselves. For example, alteplase antiviral monoclonal antibody therapies regarding(Activase, TPA) (dissolves blood clots); dornase alfa antigenic variability of circulating viral strains and the(Pulmozyme) (a recombinant DNAse I that digests ability of viruses to undergo neutralization escape wasDNA in the mucous secretions in lungs); imiglucerase reported (Cerezyme)-a recombinant glucocereborsidase for vii) Cell therapies: Cell therapy describes theGaucherûs disease, bone destruction and enlargement process of introducing new cells into tissues in order toof the liver and spleen . Factor IX (Alphanine SD, treat a disease. Several stem cell therapies are routinelyBenefix, Bebulin VH, Profilnine SD, Proplex T) used to treat disease today. Adult stem cell transplantbelonging to peptidase family S1, is one of the serine e.g. bone marrow stem cells, adult stem cell transplantproteases of the coagulation system. Deficiency of this e.g. peripheral blood stem cells and umbilical cord bloodprotein causes hemophilia B  stem cell transplant. Umbilical cord blood stem cell iii) Hormones: These chemicals transfer information transplants are less prone to rejection than either boneand instructions between cells in animals and plants. marrow or peripheral blood stem cells. The best-knownExamples include insulin (Insugen, Humulin, Novolin), stem cell therapy to date is the bone marrow transplant,human growth hormone (Ascellacrin, Crescormon), which is used to treat leukemia and other types of cancer,glucagon, growth hormone, gonadotrophins (Ovidrel) as well as various blood disorders . iv) Clotting factors: These include any factor in the Regenerative medicine using stem-cell research,blood that is essential for the blood to coagulate . tissue engineering and gene therapy is cutting-edge v) Vaccines: These are microorganisms or subunit research and it focuses on the repair, replacement andof microorganisms that can be used to stimulate regeneration of cells, tissues or organs to restoreresistance in a human to specific diseases as well as to damaged function resulting from diseases and ailments.stimulate immune response. Examples include hepatitis Stem cell-based therapies, tools and targets are ourB virus [Baraclude (Entecavir), Adefovir dipivoxil future. The big challenge for the stem cell community is(Hepsera), Lamivudine (Epivir -HBV, 3TC), Alfa therefore to facilitate the best possible interaction withInterferon (Intron A, Infergen, Roferon)], Ebola virus the population at large i.e. one stem cell world .(No commercially available Ebola vaccines are available). Stem cell treatments are a type of genetic medicineResearchers have identified a protein in infected liver that introduces new cells into damaged tissue in ordercells that is essential for hepatitis C virus replication. to treat a disease or injury. Many medical researchers
6 B.S. Sekhonbelieve that stem cell treatments have the potential to licensed, and only for topical applications . It is nowchange the face of human disease and alleviate suffering. possible to produce very large quantities of therapeuticThe ability of stem cells to self-renew and give rise to peptides with tight specifications by using the widesubsequent generations that can differentiate offered a possibilities offered by liquid phase and solid phaselarge potential to culture tissues that can replace diseased technologies, alone or in combination depending on theand damaged tissues in the body, without the risk of specific features of a given project. Moreover, manyrejection and side effects . A number of stem cell peptides currently in the preclinical or clinical stagestreatments exist, although most are still experimental contain non-natural amino acids (β-amino acids or aminoand/or costly, with the notable exception of bone marrow acids having D configuration) to make them more activetransplantation. Medical researchers anticipate one day or more stable . Selection methodologies addressingbeing able to use technologies derived from adult and protease resistance have been developed and whenembryonic stem cell research to treat cancer, Type 1 combined with methods such as pegylation antibodydiabetes mellitus, Parkinsonûs disease, Huntingtonûs Fc attachment and binding to serum albumin look likelydisease, Celiac Disease, cardiac failure, muscle damage to finally turn therapeutic peptides into a widely acceptedand neurological disorders, along with many others . drug class . viii) Antisense drugs: Antisense drug is a medicationcontaining part of the non-coding strand of messenger TechnologiesRNA (mRNA). Antisense drugs work at the genetic level PEGylationto interrupt the process by which disease-causing It is the process of covalent attachment of polyproteins are produced. Instead of attacking the bacteria (ethylene glycol) or PEG polymer chains to anotheror viruses that cause diseases, antisense drugs will literally molecule, normally a drug or therapeutic protein.throw a wrench into the portion of a cellûs genetic machinery PEGylation has been proven as a powerful new drugthat produces disease-related proteins. Among much delivery technology. Therapeutic proteins have beennew molecular therapeutics being explored for cancer modified chemically by the covalent addition of PEG, ortherapy, antisense oligonucleotides are emerging as a dextrans or other sugars, or by cross-linking to othernovel approach to cancer therapy, and used alone or in proteins with the main aims being to extend the circulationcombination with conventional treatments such as time and/or the avoidance of immunogenicity or toxicitychemotherapy and radiation, with numerous antisense of those protein drugs. The recent achievements inagents being evaluated in preclinical studies and several PEGylation processes with an emphasis on novelanticancer antisense drugs in clinical trials . One of PEG-drugs constructs, the unrealized potential ofthe treatments for genetic disorder or infections is PEGylation for non-injected routes of delivery has beenantisense therapy. When the genetic sequence of a reported. . Five PEGylated biopharmaceuticals:particular gene is known to be causative of a particular pegademase bovine (Adagen) [PEG- bovine adenosinedisease, antisense drugs hybridize with and inactivate deaminase, used to treat cross-linked severe combinedmRNA, thereby, restricting a particular gene from immunogenicity syndrome, as an alternative to boneproducing the protein for which it holds the recipe . marrow transplantation and enzyme replacement byAlthough specificity and selectivity are the key features gene therapy], pegaspargase (Oncaspar) (PEGylatedof antisense oligonucleotides, the need to target the L-asparaginase for the treatment of acute lymphoblasticright tissues and reach the nucleus remains a challenge leukemia in children who are hypersensitive to the nativeto overcome [49, 50]. unmodified form of L-asparaginase), pegylated interferon ix) Peptide therapeutics: Peptide therapeutics alfa-2a (Pegasys) or pegylated interferon alfa-2brepresents a novel class of therapeutic agents. Currently, (PegIntron) [PEGylated interferon alpha for use in theonly selected cationic antimicrobial peptides have been treatment of chronic hepatitis C and hepatitis B],
Thai J. Pharm. Sci. 34 (2010) 1-19 7pegfilgrastim (Neulasta) (PEGylated recombinant with all of their disadvantages including high buffermethionyl human granulocyte colony-stimulating factor requirements, large footprint, reuse and storage of resinfor severe cancer chemotherapy induced neutropenia), studies as well as costs.and doxorubicin HCl (Doxil) (PEGylated liposomecontaining doxorubicin for the treatment of cancer) were Bio-crystallizationcommercialized. The crystallization of biopharmaceuticals is poorly Releasable PEGylation employs customized linkers understood and is a rarely used commercial processthat reversibly bind a therapeutic moiety with polyethylene for the primary separation and purification of proteins.glycol polymers. Based on the bioconjugates of cytokines, The development of the technology base for an essentiallypeptide hormones, immunotoxins, enzymes, and reporter new biopharmaceuticals unit process operation-proteins, researchers have described both aromatic and biocrystallization is desirable. The benefits of proteinaliphatic based customized linkers that release the crystallization in biopharmaceutical processing include i)unaltered original drug under physiological conditions isolation and purification: streamlining the manufacturingand at therapeutically useful release rates . PEGylation process and making biopharmaceuticals less expensivehas become the biopharmaceutical delivery technology [62-64], ii) dosage levels-bioavailability: crystals are theof choice for intravenously administered therapeutic most concentrated form and is beneficial for drugs suchproteins . The benefits of PEGylation to a known as antibodies, which require high doses at the deliveryprotein-based biopharmaceutical versus a non-PEGylated site , iii) protein crystallization may significantly improveversion include: i) improved pharmacokinetics i.e enhanced some aspects of protein handling, and change the waysolubility, improved stability, sustained absorption, biopharmaceuticals are produced, formulated, andcontinuous biopharmaceutical action, ii) increased delivered , iv) sustained release-stability :reducedcirculation time i.e. decreased amount of protein required chemical degradation and as such may enhance drugfor therapeutic efficacy, decreased dosing frequency due efficacy over prolong period†, v) handling/processing/to optimized biodistribution, reduced renal clearance, delivery-formulation: ability to achieve high concentration,increased circulation time, iii) decreased toxicity, improved low viscosity formulation and controlled release proteinsafety profile, reduced immunogenicity, reduced proteolysis. delivery , vi) engineering to suit purpose: the abilityStructural properties of PEGylated proteins could play to control crystal shape or habit and polymorphism asan increasingly important role in developing optimal in the case of urate oxidase .therapeutic protein drugs  Therapeutic applications of proteins include Biologicals are produced under controlled conditions treatments for acute conditions, such as cancer, cardiovascularand newly generated proteins undergo complex disease and viral disease, and chronic conditions, suchpost-translational modifications. These are very sensitive as diabetes, growth hormone deficiency, haemophilia,to production conditions and minor changes can arthritis, psoriasis and Crohnûs disease. Protein crystalshave major impacts on biological activity [58-60]. have shown significant benefits in the delivery ofPost-translational modifications add to complexity, for biopharmaceuticals to achieve high concentration, lowexample, degree of glycoslyation can affect receptor viscosity formulation and controlled release proteinbinding, and pegylation can affect receptor binding delivery. The utilization of protein crystals in biopharmaceuticaland metabolic removal . applications has been reviewed . The separation and purification of biopharmaceuticals The regulatory and commercial pressures torepresents one of the most time and cost intense accurately characterize complex biopharmaceuticaldownstream operations in the manufacture of commercial molecules have lead to analysts looking beyond thebiopharmaceutical products. Separation and purification traditional tried and tested methods. Analytical techniquesof protein systems are usually achieved chromatographically include proteomic methods, mass spectrometry, 2D-PAGE,
8 B.S. SekhonCE-SDS gel method and sensitive micro/nano chromatography thaliana and others can generate many recombinant[71, 72]. proteins . The possible mechanisms by which glycosylation improves the molecular stability of proteinBiopharmaceutical manufacturing pharmaceuticals have been reported . Insulin Aspart Biopharmaceutical manufacturing is complex and (NovoLog/NovoRapid) and insulin Glargine (Lantus)variable. These may be produced from microbial cells are manufactured from recombinant DNA technology(e.g. recombinant E. coli or yeast cultures), mammalian using the yeast, Pichia pastoris. Erythropoietin-alpha iscell lines, plant cell cultures and moss plants in bioreactors a 165 amino acid glycoprotein manufactured byof various configurations, including photobioreactors recombinant DNA technology and its biological effects[73, 74]. The process of manufacturing a biopharmaceutical are the same as naturally occurring erythropoietin.product entails two major steps that are referred to as Streptokinase (Kabikinase, Streptase) is manufacturedupstream and downstream processing. Upstream by recombinant DNA technology from E. coli as aprocessing skills include those associated with the culture non-glycosylated polypeptide chain. Various aspects ofand maintenance of cells and downstream processing biopharmaceuticals development and clinical manufacturingskills included those associated with the chemical and have been reported and an outline of drug substancephysical separations necessary for the isolation and production involving upstream and downstream processpurification of the product itself, from the complex is shown in Figure 2. [79, 80]culture mixture [75, 76]. The production technologies and operations that For proteins that require glycosylation, mammalian occur in the manufacturing facility were reported .cells, fungi or the baculovirus system are chosen. The Scientists described practices applicable to thetwo most utilized yeasts are Saccharomyces cerevisiae large-scale processing of biotechnological products .and Pichia pastoris. Yeasts can produce high yields of Protein therapeutics-therapy using protein-based drugsproteins at low cost, proteins larger than 50†kD can be especially monoclonal antibodies and recombinantproduced, signal sequences can be removed, and proteins, have emerged as the hottest approach in targetingglycosylation can be carried out. The most popular system and treating a number of diseases. The demand for newfor producing recombinant mammalian glycosylated and effective biotherapeutic production has resulted inproteins is that of mammalian cells. Genetically modified new technologies associated with protein expressionanimals secrete recombinant proteins in their milk, blood and purification. In biopharmaceutical manufacturing,or urine. Similarly, transgenic plants such as Arabidopsis process development accounts for 30% of costs, upstream Cell bank vial Upstream Cell expansion Fermentation Clarification Raw materials In process testing Downstream Centrifugation Chromatography Ultrafiltration Drug substance DS release testingFigure 2 An outline of biopharmaceutical manufacturing involving upstream and downstream process for drug substance (DS) production
Thai J. Pharm. Sci. 34 (2010) 1-19 9processing for 20%. However, the highest outlay in Transgenicsbiopharmaceutical manufacturing is attributed to downstream A potentially controversial method of producingprocessing, which is responsible for a massive 40% of biopharmaceuticals involves transgenic organisms,the total costs incurred . There has been considerable particularly plants and animals that have been geneticallypressure to reduce the cost of downstream processing modified to produce drugs. Transgenic plants are anand cut the bottlenecks in the biopharmaceutical attractive platform for the production of biopharmaceuticalsproduction process. Monoclonal antibody manufacture since they offer bio-safety, lower cost of goods andpresents substantial current and upcoming challenges flexibility/scalability. One potential approach to thisto the biopharmaceutical industry . In the future, technology is the creation of a transgenic mammal thatexperts are of opinion that successful companies will can produce the biopharmaceutical in its milk (or bloodhave fewer ton-scale proteins and will look for economical or urine). The first such drug manufactured from theways to produce low-scale products. milk of a genetically-modified goat was antithrombin (ATryn). Scientists created, moss strains with non-Characterization of biopharmaceuticals immunogenic humanized glycan patterns and an overview Most commonly used spectrophotometric, of the relevant aspects for establishing moss as achromatographic, and electrophoretic methods are used production system for recombinant biopharmaceuticalsto characterize biopharmaceutical product. Mass was reported . Other technology trends includespectrometry can be used, in conjunction with protein development of efficient mammalian expression systemsand carbohydrate chemistry, to solve a variety of structural and cloning technology, though there is still some wayproblems ranging from de-novo protein sequencing, to to go before cloning becomes part of normal treatmentthe characterization of recombinant proteins, including procedure .vaccine and antibody products. Particular emphasis is The expiry for patent protections for biologics haspositioned on the identification of post-translational created a new marketplace to be exploited by genericmodifications, such as glycosylation. Glycosylation is competition . Biosimilars, or follow-on proteins, areimportant for the biological activity of proteins. The new versions of existing biopharmaceuticals whosefunctions of the glycocomponent include protein folding, patents have expired . They are produced using theprotein trafficking, protein targeting, ligand recognition, same core genetic material and are approved on theligand binding, biological activity, stability, pharmacokinetics basis that they are equal to the reference product inand immunogenicity. Scientists have applied the terms of both safety and efficacy. Biosimilars are large,MS-Mapping technique routinely to the characterization complex molecules produced by living organisms, whichof recombinant protein and glycoprotein products . are sensitive to manufacturing changes. Biosimilars isScientists highlighted the role that mass spectrometry an official term used by the European medical authorities;can and should play in the biopharmaceutical industry the US terminology is follow-on protein products. Thebeyond the presently assigned task of primary structure promise of profits from biosimilars grows greater, butanalysis . Methods and genetically engineered cells only when a number of significant market, regulatoryuseful for producing an altered N-glycosylation form of and clinical hurdles can be overcome .a target molecule was described . Analyticalultracentrifugation and field flow fractionation are two Bioprocess membrane technologyimportant biophysical methods for measuring in Bioprocessing is a crucial part of the biotechnology/characterization of therapeutic proteins in the biopharmaceutical sector and it is anticipated that withinbiopharmaceutical industry . the next five to ten years, up to 50% of all drugs in
10 B.S. Sekhondevelopment will be biopharmaceuticals; a large proportion . The technology used in biopharmaceutical filtrationof these will be recombinant protein therapeutics. and separation (the harvesting of mammalian cells toBioprocessing encompasses a wide range of techniques produce new drugs and medicines) has been reportedused in the development and manufacturing of bioscience .based medicines, (known as biopharmaceuticals orbiologics). Applications include tissue engineering Disposable systems in biopharmaceutical manufacturing(manufacturing approaches for tissue products); Disposable technologies are available for almostbiopharmaceutical formulation and delivery (novel every aspect of biopharmaceutical drug processing.mechanisms for formulating and delivering biopharmaceuticals); In the case of a recombinant biotech product, completelybioscience underpinning bioprocessing (understanding disposable manufacturing process means that each unitthe cellular and molecular processes which are predictive process operation, from fermentation through purification,of process performance and which can inform strategies to final fill-and-finish, ideally should be redesigned andfor process design and metabolic engineering); improved retooled to enable the economical single use. The benefitstools for bioprocessing (tools to accelerate bioprocess of disposable manufacturing are many; their relativedevelopment including high throughput bioprocess significance depends on the type of drug to beresearch); process modeling, improved analytics and ultra manufactured and the focus, size and resources of thescale-down systems individual biopharmaceutical company. These benefits Researchers provided an overview of recent include economy, speed to market, capacity and drugdevelopments in membrane technology, focusing on the process security .special characteristics of the membrane systems that Single-use technology in biopharmaceuticalare now used for the commercial production and manufacturingpurification of recombinant protein products [94-98]. Single-use systems are designed with partnershipsThe large-scale production of recombinant human between end-users and suppliers and users must evaluatemonoclonal antibodies demands economical purification supplier emerging technologies, sometimes with onlyprocesses with high throughputs. Membrane chromatography minimal in-house. In biopharmaceutical manufacturing,has already proven to be a powerful alternative to single-use components and systems can offer distincttraditional packed-bed chromatography in flow-through advantages over reusable, cleanable systems and willoperations, such as polishing for the removal of viruses continue to grow in importance and demand as theseand contaminants in biologics manufacturing. The benefit have potential to reduce cross contamination, andof using membrane chromatography is receiving elimination of many of the cleaning procedures .increased recognition and is now becoming a routine One challenge in using single-use technology inprocess step in large-scale biopharmaceutical manufacturing bioreactors is that not all cell lines are compatible withprocesses, as it opens the opportunity for interesting disposable bioreactors [104-107].new application areas, such as removal of impurities As most biopharmaceutical drugs are manufacturedand viruses. Some of the upcoming technologies that in small batches, the advantages offered by the usecan help to optimize time and cost of biopharmaceutical of disposables would seem especially applicable tomanufacturing have been recently reported . These this market segment. The use of disposables intechnologies include i) tangential flow filtration microfiltration biopharmaceutical manufacturing can significantly impactcapsule for cell clarification and harvest, ii) hydrophobic manufacturing process efficiency by reducing capitalinteraction membrane chromatography and iii) solutions costs, improving plant flexibility, reducing startup timefor high parvovirus retention. The disadvantages and and costs, eliminating some or all non-value addedadvantages of using Q membrane chromatography as a process steps, eliminating cross-contamination, significantlypurification unit in large-scale production were reported reducing process waste, reducing labor costs and
Thai J. Pharm. Sci. 34 (2010) 1-19 11reducing on-site quality and validation requirements. antibody (anti-lymphostat B). Scientists have discussedHowever, the development of fully disposable manufacturing the efficacy and side effects of these agents, their impactplatforms to supply all scales of biopharmaceutical on current clinical practice and future trends. .manufacturing remains in the future and is dependent The status on the use of biopharmaceuticals in theon the development of new technologies, specifically treatment of rheumatoid arthritis indicated that blockingin the areas of disposable chromatography, tangential of TNF-alpha, co-stimulation of CD28+ T-cells andflow filtration (TFF) and centrifugation by the developers depletion of CD20+ B-cells were effective ways toand producers of these technologies. However, the diminish inflammation and joint damage .long-term future of disposables looks bright as applications Viral safety is a key issue for biological andfor disposables are expanding. They will be incorporated biotechnological medicinal products. Scientists discussedinto the toolbox of technologies available to the end the most up-to-date scientific knowledge and regulatoryuser with the development of other disposable processes aspects in the areas of virus safety of recombinant[108-112]. proteins, monoclonal antibodies, plasma-derived medicinal products and advanced technology medicinal productsMiscellaneous aspects [117, 118]. New and emerging large-molecule bioactive agents Fusion proteins are successful biopharmaceuticals.delivered from stent surfaces in drug-eluting stents (DESs) Fusion proteins can be categorized into several groupsto inhibit vascular restenosis in the context of interventional according to their features. In the first group, effectorscardiology has been reported. New therapeutic agents molecules are fused to Fc domains, albumin or transferrinrepresenting proteins, nucleic acids (small interfering to extend the plasma half-life of the fusion product.RNAs and large DNA plasmids), viral delivery vectors, In the second group, toxicity is conveyed by fusionand even engineered cell therapies require specific proteins to toxins, enzymes or cytokines. The thirddelivery designs distinct from traditional smaller-molecule application, which is not yet in clinical trials, utilizesapproaches on DESs. Many of the larger-molecule and fusion partners to enable novel delivery and targetingbiopharmaceutical approaches have been reported routes. Besides some specific disadvantages, manyrecently for stent-based delivery with the challenges examples of fusion proteins suffer from the challenge ofassociated with formulating and delivering these drug immunogenicity; however, future applications with novelclasses compared to the current small-molecule drugs fusion partners will reach beyond cancer, immunology. and inflammation . The potential applications of Biologicals as a group are highly effective in the erythrocytes in drug delivery have been reviewed with atreatment of rheumatoid arthritis. Biologicals were particular stress on successful erythrocyte loading andefficacious both in treatment naïve and methotrexate- characterization of the different classes of biopharmaceuticalsrefractory patients . A recent advance in the . In recent years, recombinant therapeutic proteinsmanagement of rheumatoid arthritis is the use of produced in mammalian cells demand has increasedbiological agents which block certain key molecules dramatically and is now driving the development of ainvolved in the pathogenesis of the illness. They include variety of improvements to maximize their expression intumour necrosis factor (TNF)-blocking agents such as mammalian cells . Glycoengineered yeast can beinfliximab (Remicade), etanercept ((Enbrel) and used to produce functional full-length monoclonaladalimumab (Humira), the anti-CD 20 agent rituximab antibodies at commercially viable productivities .(Rituxan and MabThera) and CTLA-4 Ig abatacept DNA has the potential to meet the demands of emerging(Orencia). Other agents which are in development and existing diseases and of particular interest forinclude anti-IL6 tocilizumab (Actemra, RoActemra), therapeutic use is plasmid DNA that makes use of cellularanti-CD22 (epratuzumab) and monoclonal anti-BLyS machinery to express proteins or antigens. The production
12 B.S. Sekhonstages of fermentation and downstream purification using Regulatory authorities and the biopharmaceuticalDNA as a drug was reported . Scientists have industry are continuously seeking to improve methodsdescribed application of using RNAi technology to for the detection, identification, inactivation and removalincrease cellular productivity and the quality of recombinant of potentially contaminating pathogens in biotherapeutics.proteins that are produced in Chinese hamster ovary The biopharmaceutical industry has employed a(CHO) cells, the most important mammalian cell line multifaceted approach in pathogen detection, includingused in producing licensed biopharmaceuticals . the rigorous screening of blood/plasma donations;Researchers focused on the use of aqueous two-phase documented sourcing and screening of raw materials;liquid-liquid extraction technique as an option for the thorough testing of production cell substrates and celldownstream processing of biopharmaceuticals therapeutic culture harvest material during processing, and at theproteins for the purification of monoclonal antibodies, stage of a final purified drug substance; and the evaluationgrowth factors and hormones . Monoclonal antibodies of microbe clearance during purification operations .have become major therapeutic drugs for the treatment High-tech bioreactor-derived bioactive phytomoleculesof a number of diseases, thanks to a remarkable molecular and biopharmaceuticals hold the prospect of providingengineering. The success of the first generation of permanent remedies for improving human well-beingmonoclonal antibodies opens the way to new challenges .such as antibody functional optimization, better controlof unwanted side effects, or low cost production at an Some specific uses of biopharmaceuticalsindustrial scale. A new generation of antibodies is now Erythropoietin: Erythropoietin is the hormoneemerging and one can already foresee the future: responsible for inducing red blood cell production byoligoclonal approaches based on the use of specific the bodyûs bone marrow. The most common use is inantibodies cocktails, selection of eligible patients, and people with anemia (low blood count) related to kidneyantibody production at low costs . dysfunction. Drugs such as epoietin alfa (Epogen, Twenty five percent of women who have breast Procrit) and darbepoetin alfa (Aranesp) increased thecancer express a growth receptor called HER2. Breast production of red blood cells. They are used to treatcancer with this receptor expressed grow exceptionally anaemia associated with chronic kidney failure, cancerfast compared to other types. A genetically engineered chemotherapy, and antiretroviral HIV therapy .monoclonal antibody trastuzumab (Herceptin) by Genentech, Erythropoietin plays an important role in the brainûsInc, is a drug designed specifically to block the activity response to neuronal injury and is also involved in theof the HER2 receptor. It does not work in patients who wound healing process [132-134].do not express the HER2 receptor, but it works Interferon-α : Imatinib (Gleevec) is a 2-phenylamino-exceptionally in those patients who do express it. pyrimidine derivative that functions as a specific inhibitorScientists consider it as the ideal example of a truly of a number of tyrosine kinase (TK) enzymes. It occupiestargeted biopharmaceutical . the TK active site, leading to a decrease in activity. New antibody discovery tools have increased the IMATINIB is used in chronic myelogenous leukemia,speed and precision with which potent neutralizing human gastrointestinal stromal tumors and a number of otherantiviral monoclonal antibodies (mAbs) can be identified. malignancies .As longstanding barriers to antiviral mAb development, Interferon-β : This is used for treatment of relapsingsuch as antigenic variability of circulating viral strains multiple sclerosis .and the ability of viruses to undergo neutralization escape, Monoclonal antibody: The newest monoclonalare being overcome, deeper insight into the mechanisms antibody approved for the treatment of rheumatoid arthritisof mAb action and engineering of effector functions are is rituximab (Rituxan). Like infliximab (Remicade), it is aalso improving the efficacy of antiviral mAbs . chimeric mouse/human monoclonal antibody that is given
Thai J. Pharm. Sci. 34 (2010) 1-19 13by intravenous infusion. Unlike Remicade, it attacks in healthy volunteers .the B cells as opposed to TNF. Several new monoclonal Glucocerebrosidase: This is used for treatment ofantibodies are in the development stage to treat Gaucherûs disease which is the most common glycolipidrheumatoid arthritis and other conditions. Trends in storage disorder, characterized by storage of thebenchmarks for various types of monoclonal antibodies, glycolipid, glucocerebroside in the liver, spleen, andwith an emphasis on that study as anticancer and marrow. It is caused by a hereditary deficiency ofimmunological therapeutics were reported . the enzyme glucocerebrosidase (also known as acidResearchers provided an overview of the upstream β-glucosidase). Enzyme replacement therapy, withtechnologies used in the industrial production of glucocerebrosidase purified from human placentae,therapeutic monoclonal antibodies based on the was introduced in 1991. Recombinant human glucocere-cultivation of mammalian cells. They reviewed the cell brosidase, produced by Chinese hamster ovary cells inlines currently employed in commercial production and tissue culture, became available in 1994 and has replacedthe methods of constructing and isolating production the placenta-derived product. These therapies haveclones, followed with a review of the most current revolutionized the care of patients with type 1 Gaucherûsmethods of commercial scale production and their disease, reversing many of the pathological consequencesassociated technologies . of this disease, and preventing further progression. The recent knowledge on the use, effectiveness Furthermore, they have served as a model for theand safety of monoclonal antibodies in multiple sclerosis treatment of other lysosomal storage diseases and inborntreatment has been reported . Natalizumab (Tysabri, errors of metabolism [143, 144]. The relatively higha humanized monoclonal antibody) is a prescription prevalence of this disease within an ethnic group ismedication approved for patients with relapsing forms believed to reflect a selective advantage. Treatment withof multiple sclerosis, and is now indicated for appropriate enzyme replacement therapy is safe and effective inpatients with Crohnûs disease. Tysabri binds to ameliorating the primary symptoms of the disease, yetthe çalpha4beta1é integrins of leukocytes, and blocks there have been reports that some patients on enzymeattachment to cerebral endothelial cells, thus reducing replacement therapy have developed type 2 diabetes orinflammation at the blood-brain barrier. The mandatory metabolic syndrome, malignancies and central nervousguidelines for natalizumab use in the treatment of system disorders .multiple sclerosis were also reported. Natalizumabrecently joined glatiramer acetate (Copaxone, or Conclusion and perspectivescopolymer 1) and interferon beta-1b (Betaseron) as Biopharmaceuticals are medical drugs derived froman approved therapy for controlling relapsing multiple biological sources and especially one produced bysclerosis . biotechnology. They are therapeutic proteins, cellular Colony stimulating factors; Colony-stimulating products, gene therapy products, vaccines and plasmafactors are medications used to increase the number of blood product derivatives. Leading biopharmaceuticalWBCs. Recombinant human granulocyte colony-stimulating products include erythropoietins, insulins, and monoclonalfactor (G-CSF) has been used for treatment of febrile antibodies, and biopharmaceutical companies are turningneutropenia in systemic lupus erythematosus (SLE) and their focus on long-term conditions such as cardiovascularother systemic rheumatic diseases. However, scientists disease, diabetes and asthma. In light of limitations ofsuggested that G-CSF therapy should be used with cost and lack of long-term safety and efficacy data,considerable caution in patients with SLE . AVI-014 newer agents for the time being are recommended foris an egg white-derived G-CSF and has safety, use as second- or third-line agents in patients withpharmacokinetic, and pharmacodynamic properties active rheumatoid arthritis. One alternative approach maycomparable to Neupogen (Filgrastim) at an equal dose be to limit the use in patients who can afford it, and
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